PROJECT SUMMARY The proposed studies develop a novel, site-specific, and sequence-specfic DNA cross-linking reaction as a signal output for the detection of nucleic acid sequence and structure in bioanalytical applications. A nanopore will be used for single-molecule detection of cross-linked DNA. We have selected the detection of single-nucleotide polymorphisms (SNPs) as a test-bed to demonstrate the general utility of this approach. Many assays use noncovalent, reversible hybridization of a probe to target for detection of nucleic acid sequence. Our approach employing sequence-specific covalent cross-linking to the target strand is novel. In this application, we propose to develop sequence-specific DNA cross-linking as a new single-molecule signal-output for use with nucleic acid-based sensors and probes. The proposed work builds upon our extensive preliminary data including: (i) sequence-selective cross-link formation in DNA duplexes containing a reactive Ap site (Gates), (ii) use of nanopore technology for the bioanalytical characterization of nucleic acid sequence and structure (Gu), and (iii) use of nanopore technology for the single-molecule detection of DNA duplexes containing interstrand cross-links (Gates and Gu). We hypothesize that sequence-selective cross-linking reactions can be used for the sensitive detection of disease-relevant nucleic acid sequences. Experiments to test this hypothesis are divided into the following Three Specific Aims: (1) Use biochemical methods to characterize and optimize the ability of Ap- containing probes to detect disease-relevant DNA sequences via sequence-specific cross-linking reactions. (2) Develop and optimize methods for the quantitative detection of cross-linked DNA duplexes in the ?-hemolysin (HL) protein nanopore, including the use of Ap-containing probe strands that optimize capture and unzipping in the nanopore, engineered protein pores, and barcoded probe strands. (3) Apply the methods developed in Aims 1 and 2 to the detection of disease-relevant DNA sequences in human cell lines. The outcomes of our studies include the characterization of sequence-selective cross-linking reactions for the accurate detection of polymorphisms in disease-relevant human DNA sequences and characterization of the resulting cross-linked duplexes using nanopore technology. The long-term goal of our research is to exploit new sequence-specific cross-linking reactions and the unmistakable current signature of cross-linked duplexes in the ?- HL nanopore to develop a single-molecule detection strategy that can provide a robust signal output to any hybridization-based analytical strategy.